Method for treatment of pulmonary fibrosis

ABSTRACT

Methods and compositions of the invention relate to the treatment of pulmonary fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/999,389, filed Feb. 20, 2014, which claims the benefit of andpriority to U.S. provisional Application Ser. No. 61/767,057, filed Feb.20, 2013, the entire disclosure of each of which is incorporated byreference in their entireties.

FIELD OF THE INVENTION

Methods and compositions of the invention relate compositions andmethods for treatment of pulmonary fibrosis.

BACKGROUND OF THE INVENTION

Pulmonary fibrosis is the formation or development of excess fibrousconnective tissue (fibrosis) in the lungs. Approximately 5 millionpeople worldwide are affected by pulmonary fibrosis. Pulmonary fibrosisis a fatal disease in which the uncontrolled deposition of extracellularmatrix leads to progressive loss of lung function.

SUMMARY OF THE INVENTION

Aspects of the invention relate to novel approaches to treat pulmonaryfibrosis.

Aspects of the invention relate to compositions, methods of using andmethods of manufacturing compositions capable of treating pulmonaryfibrosis.

Other aspects of the invention relate to methods of treating a subjectin need thereof. In some embodiments, the method comprises the step ofobtaining a composition for parenteral administration, the compositioncomprising a compound in an acceptable pharmaceutical carrier andadministering the composition to a subject in need thereof.

In some embodiments, the compound can be one ofgalacto-rhamnogalacturonate (GRG), galactoarabino-rhamnogalacturonate(GA-RG), galactomannan (GM), or a combination of any of the foregoing.

In some embodiments, the compound can be a polysaccharide chemicallydefined as galacto-rhamnogalacturaonate (GRG), a selectivelydepolymerized branched heteropolymer whose backbone is predominantlycomprised of 1,4-linked galacturonic acid (GalA) moieties, with a lesserbackbone composition of alternating 1,4-linked GalA and 1,2-linkedrhamnose (Rha), which in-turn is linked to any number of side chains,including predominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose(Ara) residues. Other side chain minor constituents may include xylose(Xyl), glucose (Glu), and fucose (Fuc) or combinations thereof.

In some embodiments, the GRG compound can be produced as described inU.S. Patent Application Publication No. 2008/0107622, now U.S. Pat. No.8,236,780, which is incorporated expressly by reference for allpurposes.

In some embodiments, the GRG compound can be produced as described inU.S. Pat. Nos. 8,128,966, 8,187,624, U.S. Patent Application PublicationNos 2012/0315309 and 2012/0309711 which are incorporated expressly byreference for all purposes.

In some embodiments, the compound can be a polysaccharide chemicallydefined as galactoarabino-rhamnogalacturonate (GA-RG), a selectivelydepolymerized, branched heteropolymer whose backbone is predominantlycomprised of 1,4-linked galacturonic acid (GalA) moieties, with a lesserbackbone composition of alternating 1,4-linked GalA and 1,2-linkedrhamnose (Rha), which in-turn is linked to any number of side chains,including predominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose(Ara) residues. Other side chain minor constituents may include xylose(Xyl), glucose (Glu), and fucose (Fuc) or combinations thereof.

In some embodiments, the GA-RG compound can be produced as described inInternational Patent Application PCT/US12/55311, and U.S. PatentApplication Number US 2013-0261078, which is incorporated expressly byreference for all purposes. In some embodiments, the compound can be agalactomannan (GM) polysaccharide composition produced as described inU.S. Pat. No. 8,236,780 and U.S. Patent Application Publication No. US2011/0077217 which are incorporated expressly by reference in theirentireties for all purposes.

Some aspects of the invention relate to a method comprising obtaining acomposition for parenteral or enteral administration comprising agalacto-rhamnogalacturonate in a pharmaceutical acceptable carrier, andadministering to a subject in need thereof an effective dose of thecomposition that results in at least one of the following: at least 5%reduction of lung edema, at least 5% reduction of lung pathologyseverity scores associated with lung fibrosis, at least 5% reduction oflung tissue hydroxyproline accumulation, at least 5% reduction ofexpression of pro-inflammatory proteins, at least 5% reduction ofexpression of fibrogenic proteins. In some embodiments, theadministration of the effective dose results in at least 5% improvementof pulmonary function tests including forced vital capacities or oxygendiffusion. In some embodiments, the subject in need has at least one ofthe following: a primary or secondary lung fibrotic disease.

In some embodiments, the effective dose of galacto-rhamnogalacturonatecan be equivalent to an animal dose of about 120 mg/kg. In someembodiments, the effective dose of galacto-rhamnogalacturonate can beequivalent to an animal dose of 10 mg/kg to 180 mg/kg. In someembodiments, the effective dose can be given once, twice or three timesweekly.

In some embodiments, the administration of the effective dose results inat least 5% reduction of expression of pro-inflammatory proteins. Insome embodiments, the pro-inflammatory proteins can comprise TGF-beta,IL-6, IL-8, IL-13, IP-10, osteopontin, TNF-alpha, CXCL-9/10, VEGF or anycombination of the foregoing.

In some embodiments, the administration of the effective dose results inat least 5% reduction of expression of fibrogenic proteins. In someembodiments, the fibrogenic proteins can comprise collagen, elastin or acombination thereof.

In some embodiments, the galacto-rhamnogalacturonate can comprise a1,4-linked galacturonic acid (GalA) and methyl galacturonate (MeGalA)residues backbone linked to branched heteropolymers of alternatingoligomers of α-1,2 linked rhamnose and α-1,4-linked GalA residues, therhamnose residues carrying a primary branching of oligomers of1,4-β-D-galactose residues.

In some embodiments, the galacto-rhamnogalacturonate can comprise a1,4-linked galacturonic acid (GalA) residues backbone linked to branchedheteropolymers of alternating oligomers of α-1,2 linked rhamnose andα-1,4-linked GalA residues, the rhamnose residues carrying a primarybranching of oligomers of 1,4-β-D-galactose residues.

In some embodiments, the galacto-rhamnogalacturonate can furthercomprise xylose, glucose, fucose residues or combination thereof.

In some embodiments, the galacto-rhamnogalacturonate can besubstantially free of 1,5-α-L-Ara residues.

In some embodiments, the galacto-rhamnogalacturonate is agalactoarabino-rhamnogalacturonate, comprising a 1,4-linked galacturonicacid (GalA) and methyl galacturonate (MeGalA) residues backbone linkedto branched heteropolymers of alternating oligomers of α-1,2 linkedrhamnose and α-1,4-linked GalA residues, the rhamnose residues carryinga primary branching of oligomers of 1,4-β-D-galactose residues,1,5-α-L-arabinose residues, or combinations thereof.

In some embodiments, the 1,4-β-D-galactose and 1,5-α-L-arabinoseresidues are present in a 2:1 or a 3:1 ratio.

In some embodiments, the 1,4-β-D-galactose residues, the1,5-α-L-arabinose residues or combination thereof can represent at least10 molar percent of the total molar carbohydrates.

In some embodiments, the galacto-rhamnogalacturonate can have an averagemolecular weight ranging from 5 kDa to 55 kDa as determined by SEC-RIand/or the SEC-MALLS methods.

In some embodiments, the galacto-rhamnogalacturonate can have an averagemolecular weight ranging from 2 kDa to 80 kDa as determined by SEC-RIand/or the SEC-MALLS methods.

In some embodiments, the galacto-rhamnogalacturonate can an averagemolecular weight ranging from 20 kDa to 70 kDa as determined by SEC-RIand/or the SEC-MALLS methods.

In some aspects, the method comprises obtaining a composition forparenteral or enteral administration comprising a galactomannanpolysaccharide in a pharmaceutical acceptable carrier, and administeringto a subject in need thereof an effective dose of the composition thatresults in at least one of the following: at least 5% reduction of lungoedema, at least 5% reduction of lung pathology associated with lungfibrosis, at least at least 5% reduction of hydroxyproline accumulation,at least 5% reduction of expression of pro-inflammatory proteins, atleast 5% reduction of expression of fibrogenic proteins, wherein thesubject in need thereof has at least one of the following: a primary orsecondary lung fibrotic disease. In some embodiments, the administrationof the effective dose results in at least 5% improvement of pulmonaryfunction tests including forced vital capacities or oxygen diffusion.

In some embodiments, the effective dose of the galactomannanpolysaccharide is equivalent to an animal dose of about 60 mg/kg. Insome embodiments, the effective dose of the galactomannan polysaccharideis equivalent to an animal dose of 30 mg/kg to 180 mg/kg. In someembodiments, the effective dose can be given once, twice or three timesweekly.

In some embodiments, in the step of obtaining, the galactomannanpolysaccharide has a mannose to galactose ratio ranging from 1:1 to 1:4.

In some embodiments, in the step of obtaining, the galactomannanpolysaccharide has a mannose to galactose ratio of 1.7:1.

In some embodiments, in the step of obtaining, the galactomannanpolysaccharide has a molecular weight of about 48,000 D.

In some embodiments, in the step of obtaining, the galactomannanpolysaccharide has average molecular weight ranging from 4,000 D to60,000D.

In some embodiments, the administration of the effective dose results inat least 5% reduction of expression of pro-inflammatory proteins. Insome embodiments, the pro-inflammatory proteins can comprise TGF-beta,IL-6, IL-8, IL-13, IP-10, osteopontin, TNF-alpha, CXCL-9/10, VEGF or anycombination of the foregoing.

In some embodiments, the administration of the effective dose results inat least 5% reduction of expression of fibrogenic proteins. In someembodiments, the fibrogenic proteins can comprise collagen, elastin or acombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals throughout the several views. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the present invention.

FIGS. 1A and 1B depicts the change in animal weight in the mouse modelof bleomycin-induced pulmonary fibrosis with early treatment withGR-MD-02 and GM-CT-01. FIG. 1A shows the daily weight change of miceexpressed as mean and standard error of the mean. FIG. 1B shows thepercent change in weight for each treatment group as calculated usingthe area under the curve (AUC).

FIG. 2 is a graph showing the mean lung wet weight on day 21 in treatedand control groups, according to one embodiment of the invention.

FIG. 3 is a graph showing the mean total lung hydroxyproline content(expressed as a percent of saline treated mice) in mice on day 21 intreated and control groups, according to one embodiment of theinvention.

FIG. 4 is a graph showing the modified Ashcroft lung fibrosis indexscores in treated and control groups, according to one embodiment of theinvention.

FIG. 5 is a graph showing the general pulmonary fibrosis scores ontreated and control groups, according to one embodiment of theinvention.

FIG. 6 is a graph showing the inflammation scores on treated and controlgroups, according to one embodiment of the invention.

FIG. 7 are representative photomicrographs with trichrome from treatedand control groups, according to one embodiment of the invention.

FIG. 8 are representative photomicrographs with H & E from treated andcontrol groups, according to one embodiment of the invention.

FIG. 9 is a graph showing the weights of the lung of the control groups,vehicle treated group, and treated groups.

FIG. 10 is a graph showing the lung hydroxyproline (expressed as apercent of saline treated mice) of the control groups, vehicle treatedgroup, and treated groups.

FIG. 11 is a graph showing the Aschroft fibrosis index score from ahistology assessment of the control groups, vehicle treated group, andtreated groups.

FIG. 12 is a graph showing the general fibrosis score from a histologyassessment of the control groups, vehicle treated group, and treatedgroups.

FIG. 13 is a graph showing the inflammation score from a histologyassessment of the control groups, vehicle treated group, and treatedgroups.

FIG. 14 are representative photomicrographs with trichrome from treatedand control groups, according to one embodiment of the invention.

FIG. 15 are representative photomicrographs with H & E from treated andcontrol groups, according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein;however, it is to be understood that the disclosed embodiments aremerely illustrative of the invention that may be embodied in variousforms. In addition, each of the examples given in connection with thevarious embodiments of the invention is intended to be illustrative, andnot restrictive. Further, the figures are not necessarily to scale, somefeatures may be exaggerated to show details of particular components. Inaddition, any measurements, specifications and the like shown in thefigures are intended to be illustrative, and not restrictive. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting, but merely as a representative basis forteaching one skilled in the art to variously employ the presentinvention.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application.

Unless otherwise specified, all percentages expressed herein areweight/weight.

Aspects of the invention relate to novel approaches to treat pulmonaryfibrosis using a complex carbohydrate pharmaceutical product or otheragents described herein. In some embodiments, the methods disclosedherein relate to the treatment of a subject having a primary orsecondary lung fibrotic disease.

Galectins

Galectins (also known as galaptins or S-lectin) are a family of lectinswhich bind beta-galactoside. Galectin as general name was proposed in1994 for a family of animal lectins (Barondes, S.H., et al.: Galectins:a family of animal beta-galactoside-binding lectins. Cell 76, 597-598,1994), The family is defined by having at least one characteristiccarbohydrate recognition domain (CRD) with an affinity forbeta-galactosides and sharing certain sequence elements. Within the samepeptide chain, some galectins have a CRD with only a few additionalamino acids, whereas others have two CRDs joined by a link peptide, andone (galectin-3) has one CRD joined to a different type of domain. Thegalectin carbohydrate recognition domain (CRD) is a beta-sandwich ofabout 135 amino acids. The two sheets are slightly bent with 6 strandsforming the concave side and 5 strands forming the convex side. Theconcave side forms a groove in which carbohydrate is bound (Leffler H,Carlsson S, Hedlund M, Qian Y, Poirier F (2004). “Introduction togalectins”. Glycoconj. J. 19 (7-9): 433-40).

A wide variety of biological phenomena have been shown to be related togalectins, e.g., development, differentiation, morphogenesis, tumormetastasis, apoptosis, RNA splicing, etc. However, relatively little isknown about the mechanism by which galectins exert these functions,particularly in terms of carbohydrate recognition.

Generally, the carbohydrate domain binds to galactose residuesassociated with glycoproteins. At least fifteen mammalian galectinproteins have been identified which have one or two carbohydrate domainin tandem.

Galectin proteins are found in the intracellular space where they havebeen assigned a number of functions and are secreted into theextracellular space. In the extracellular space, galectin proteins canhave multiple functions including promoting interactions betweenglycoproteins that may lead to reduced function, or enhanced functions,or in the case of integral membrane glycoprotein receptors, modificationof cellular signaling (Sato et al “Galectins as danger signals inhost-pathogen and host-tumor interactions: new members of the growinggroup of “Alarmins.” In “Galectins,” (Klyosov, et al eds.), John Wileyand Sons, 115-145, 2008, Liu et al “Galectins in acute and chronicinflammation,” Ann. N. Y. Acad. Sci. 1253: 80-91, 2012). Galectinproteins in the extracellular space can additionally promote cell-celland cell matrix interactions (Wang et al., “Nuclear and cytoplasmiclocalization of galectin-1 and galectin-3 and their roles in pre-mRNAsplicing.” In “Galectins” (Klyosov et al eds.), John Wiley and Sons,87-95, 2008).

Galectins have been shown to have domains which promotehomodimerization. Thus, galectins are capable of acting as a “molecularglue” of sorts between glycoproteins. Galectins are found in multiplecellular compartments, including the nucleus and cytoplasm, and aresecreted into the extracellular space where they interact with cellsurface and extracellular matrix glycoproteins. The mechanism ofmolecular interactions can depend on the localization. While galectinscan interact with glycoproteins in the extracellular space, theinteractions of galectin with other proteins in the intracellular spacegenerally occurs via protein domains. In the extracellular space theassociation of cell surface receptors may increase or decrease receptorsignaling or the ability to interact with ligands. Galectin proteins aremarkedly increased in a number of animal and human disease states,including but not limited to diseases associated with inflammation,fibrosis, autoimmunity, and neoplasia. Galectins have been directlyimplicated in the disease pathogenesis, as described below. For example,diseases states that may be dependent on galectins include, but are notlimited to, acute and chronic inflammation, allergic disorders, asthma,dermatitis, autoimmune disease, inflammatory and degenerative arthritis,immune-mediated neurological disease, fibrosis of multiple organs(including but not limited to liver, lung, kidney, pancreas, and heart),inflammatory bowel disease, atherosclerosis, heart failure, ocularinflammatory disease, a large variety of cancers.

In addition to disease states, galectins are important regulatorymolecules in modulating the response of immune cells to vaccination,exogenous pathogens and cancer cells.

One of skill in the art will appreciate that compounds that can bind togalectins and/or alter galectin's affinity for glycoproteins, reducehetero- or homo-typic interactions between galectins, or otherwise alterthe function, synthesis, or metabolism of galectin proteins may haveimportant therapeutic effects in galectin-dependent diseases.

Galectins show an affinity for galactose residues attached to otherorganic compounds, such as in lactose [(β-D-Galactosido)-D-glucose],N-acetyl-lactosamine, poly-N-acetyllactosamine, galactomannans,fragments of pectins, as well as other galactose containing compounds.It should be noted that galactose by itself does not bind to galectins,or binds so weakly that the binding can hardly be detected.

Pectin and modified pectin have been shown to bind to galectin proteinspresumably on the basis of containing galactose residues that arepresented in the context of a macromolecule, in this case a complexcarbohydrate rather than a glycoprotein in the case of animal cells.

Galectin proteins have been shown to be markedly increased ininflammation, fibrotic disorders, and neoplasia (Ito et al. “Galectin-1as a potent target for cancer therapy: role in the tumormicroenvironment”, Cancer Metastasis Rev. PMID: 22706847 (2012),Nangia-Makker et al. Galectin-3 binding and metastasis,” Methods MolBiol. 878: 251-266, 2012, Canesin et al. Galectin-3 expression isassociated with bladder cancer progression and clinical outcome,” TumourBiol. 31: 277-285, 2010, Wanninger et al. “Systemic and hepatic veingalectin-3 are increased in patients with alcoholic liver cirrhosis andnegatively correlate with liver function,” Cytokine. 55: 435-40, 2011.Moreover, experiments have shown that galectins, particularly galectin-1and galectin-3, are directly involved in the pathogenesis of theseclasses of disease (Toussaint et al., “Galectin-1, a gene preferentiallyexpressed at the tumor margin, promotes glioblastoma cell invasion.”,Mol Cancer. 11:32, 2012, Liu et al 2012, Newlaczyl et al., “Galectin-3—ajack-of-all-trades in cancer,” Cancer Lett. 313: 123-128, 2011, Banh etal., “Tumor galectin-1 mediates tumor growth and metastasis throughregulation of T-cell apoptosis,” Cancer Res. 71: 4423-31, 2011, Lefrancet al., “Galectin-1 mediated biochemical controls of melanoma and gliomaaggressive behavior,” World J. Biol. Chem. 2: 193-201, 2011, Forsman etal., “Galectin 3 aggravates joint inflammation and destruction inantigen-induced arthritis,” Arthritis Reum. 63: 445-454, 2011, de Boeret al., “Galectin-3 in cardiac remodeling and heart failure,” Curr.Heart Fail. Rep. 7, 1-8, 2010, Ueland et al., “Galectin-3 in heartfailure: high levels are associated with all-cause mortality,” Int JCardiol. 150: 361-364, 2011, Ohshima et al., “Galectin 3 and its bindingprotein in rheumatoid arthritis,” Arthritis Rheum. 48: 2788-2795, 2003).

The term “effective dose” means the amount of the GRG, GA-RG, GM,compounds described herein when administered as a parental dose to ananimal or human result in at least 5% decrease of a chosen molecularmarker.

In some embodiments, the effective dose of galacto-rhamnogalacturonatecan be equivalent to an animal dose of about 120 mg/kg. In someembodiments, the effective dose of galacto-rhamnogalacturonate can beequivalent to an animal dose of 10 mg/kg to 180 mg/kg. In someembodiments, the effective dose of the galactomannan polysaccharide isequivalent to an animal dose of about 60 mg/kg. In some embodiments, theeffective dose of the galactomannan polysaccharide is equivalent to ananimal dose of 30 mg/kg to 180 mg/kg. In some embodiments, the effectivedose can be given once, twice or three times weekly.

The term “efficacy” means demonstrating an improvement of lung fibrosisof at least 10% using treatment with the GRG, GA-RG, or GM compoundsdescribed herein as compared to treatment with vehicle-treated subject.

Other aspects of the invention relate to methods of treating a subjectin need thereof. In some embodiments, the method comprises the step ofobtaining a composition for intravenous or subcutaneous administrationcomprising a compound in an acceptable pharmaceutical carrier.

In some embodiments, the compound is a polysaccharide chemically definedas galacto-rhamnogalacturonate (GRG), a selectively depolymerized,branched heteropolymer whose backbone is predominantly comprised of1,4-linked galacturonic acid (GalA) moieties, with a lesser backbonecomposition of alternating 1,4-linked GalA and 1,2-linked rhamnose(Rha), which in-turn is linked to any number of side chains, includingpredominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose (Ara)residues. Other side chain minor constituents may include xylose (Xyl),glucose (Glu), and fucose (Fuc) or combinations thereof.

In some embodiments, the GRG compound is produced as described in U.S.Pat. No. 8,236,780, which are incorporated expressly by reference forall purposes.

In some embodiments, the GRG compound can be produced as described inU.S. Pat. Nos. 8,128,966, 8,187,624, U.S. Patent Application PublicationNos 2012/0315309 and 2012/0309711 which are incorporated expressly byreference for all purposes.

In some embodiments, the compound is a polysaccharide chemically definedas galactoarabino-rhamnogalacturonate (GA-RG), a selectivelydepolymerized, branched heteropolymer whose backbone is predominantlycomprised of 1,4-linked galacturonic acid (GalA) moieties, with a lesserbackbone composition of alternating 1,4-linked GalA and 1,2-linkedrhamnose (Rha), which in-turn is linked to any number of side chains,including predominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose(Ara) residues. Other side chain minor constituents may include xylose(Xyl), glucose (Glu), and fucose (Fuc) or combinations thereof.

In some embodiments, the GA-RG compound is produced as described inInternational Patent Application PCT/US12/55311, which is incorporatedexpressly by reference for all purposes.

In some embodiments, the molar percent of the 1,4-b-D-Gal and1,5-a-L-Ara residues in the GA-RG compound of the present invention is21.5% with a molar ratio of 3:1 of 1,4-b-D-Gal to 1,5-a-L-Ara.

In some embodiments, the compound is a polysaccharide chemically definedas galactoarabino-rhamnogalacturonate (GA-RG), with a molecular weightrange of 20,000 to 70,000 Daltons as determined by SEC-RI method.

In some embodiments, the compound is a galactomannan (GM) polysaccharidecomposition produced as described in U.S. Patent ApplicationUS20110077217, incorporated expressly by reference in its entirety forall purposes.

In some embodiments, the average molecular weight of the GM compound isapproximately 4,000 and 60,000 Da, as determined by the SEC-MALLSmethod.

In some embodiments, the complex carbohydrate compounds described hereincan target secreted and membrane-associated galectins, for examplegalectin-3.

In some embodiments, if an inducer of fibrosis, such bleomycinadministered by intra-tracheal instillation, causes formation of thebiochemical markers, but a concurrent administration of the inducer anda suitable complex carbohydrate compounds including but not limited toGM, GR, or GA-RG, does not lead to formation of the same marker, orleads to formation of the same marker but in a reduced amount, thepolysaccharide prevents or slows down fibrosis.

Some aspects of the invention relate to a method of treating a patientat risk of pulmonary fibrosis. The method comprises administering to apatient a pharmaceutical composition comprising the carbohydratecompounds described herein in an amount sufficient to reduce the risk ofdeveloping pulmonary fibrosis. Other aspects of the invention relate tomethod of treating pulmonary fibrosis. The method comprisesadministering to a patient a pharmaceutical composition comprising thecarbohydrate compounds described herein in an amount sufficient toreduce significantly one or more of the following: lung oedema, lungpathology associated with lung fibrosis, hydroxyproline accumulation,and expression of pro-inflammatory proteins, such as TGF-beta, IL-6,IL-8, IL-13, IP-10, osteopontin, TNF-alpha, CXCL-9/10, VEGF, andfibrogenic proteins, such as collagen and, elastin.

In some embodiments, the administration of the compound results in atleast 5% reduction of lung oedema, at least 5% reduction of lungpathology associated with lung fibrosis, at least at least 5% reductionof hydroxyproline accumulation, at least 5% reduction of expression ofpro-inflammatory proteins, at least 5% reduction of expression offibrogenic proteins, wherein the subject in need thereof has at leastone of the following: a primary or secondary lung fibrotic disease. Insome embodiments, the administration of the effective dose results in atleast 5% improvement of pulmonary function tests including forced vitalcapacities or oxygen diffusion.

EXAMPLE Mouse Model Example 1 Early Treatment of Pulmonary FibrosisInduced by Bleomycin in C57BI/6 Mice

Instillation of bleomycin in the trachea induces changes similar todiffuse pulmonary fibrosis and/or fibrosing alveolitis in humans. Thereis an increase in deposition and net synthesis of collagen in the lungwith this model.

This mouse model of pulmonary fibrosis is used extensively inpre-clinical evaluation of drugs, as indicated in a review that reportson 240 individual studies (Int J of Biochem & Cell Biology 40 (2008)362-382)

This model can be used to evaluate therapy started in the inflammatoryphase of the belomycin insult (started<7 day after bleomycin) or afterthe inflammatory phase and during fibrogenesis (started>7 day afterbleomycin). In this example, early therapy was evaluated.

The GM (GM-CT-01) and GA-RG (GR-MD-02) compounds were evaluated aspreventative therapy in this mouse model.

GM-CT-01 is a galactomannan (GM) isolated from seeds of Cyamopsistetragonoloba, or Guar gum, and subjected to a controlled partialchemical degradation. A backbone of the galactomannan is composed of(1→4)-linked β-D-mannopyranosyl units, to which singleα-D-galactopyranosyl is attached by (1→6)-linkage. Chemical names of thegalactomannan are 1,4-β-D-Galactomannan, or[(1→6)-α-D-galacto-(1→4)-β-D-mannan]. The average repeating unit ofGM-CT-01 consists of seventeen β-D-Man residues and ten α-D-Gal residues(Man/Gal ratio is 1.7), and an average polymeric molecule containsapproximately 12 of such repeating units (for the average molecularweight ranging from 42,000 Da through 60,000 Da).

GR-MD-02 is a galactoarabino-rhamnogalacturonate (GARG), a selectivelydepolymerized, branched heteropolymer whose backbone is predominantlycomprised of 1,4-linked galacturonic acid (GalA) moieties, with a lesserbackbone composition of alternating 1,4-linked GalA and 1,2-linkedrhamnose (Rha), which in-turn is linked to any number of side chains,including predominantly 1,4-b-D-galactose (Gal) and 1,5-a-L-arabinose(Ara) residues. Other side chain minor constituents may include xylose(Xyl), glucose (Glu), and fucose (Fuc) or combinations thereof. TheGR-MD-02 has an average molecular weight ranging from 20,000 Da to70,000 Da.

Pulmonary fibrosis was induced in animals using intra-trachealinstillation of bleomycin at 2.25 U/kg. Animals were treated using a GMcompound (GM-CT-01) or GA-RG compound (GR-MD-02) delivered byintravenous infusion as described in Table 1.

TABLE 1 Bleomycin Group Number of Dose Treatment Test Article NumberAnimals (Day 0) (IV) Dosing 1 10 Saline — — 2 12 2.25 U/kg Vehicle QDDays: −1, 2, 6, 9, 13, 16, 20 3 12 2.25 U/kg GM-CT-01 QD 120 mg/kg Days:−1, 2, 6, 9, 13, 16, 20 Days 4 12 2.25 U/kg GR-MD-02 Days: −1, 2,  60mg/kg 6, 9, 13, 16, 20

Animals were evaluated for their weight loss (FIG. 1A-B). While thereappeared to be a qualitative difference in weight loss between somegroups, quantitative analysis of area under the curve showed nodifference between groups.

Animals were euthanized on Day 21 and the lungs were weighed. Data shownin FIG. 2 represent group means and SEM. Statistical comparison amongall groups was performed using one-way ANOVA with Newman-Keuls multiplecomparison test. Both the GM-CT-01 and the GR-MD-02 compounds were shownto significantly reduce lung weight in comparison to vehicle-treatedmice.

After euthanasia, the total lung hydroxyproline content was measured.Data shown in FIG. 3 represent group mean and SEM and expressed asmicrogram of hydroxyproline per mg of lung tissue. Statisticalcomparison to the “vehicle+Bleomycin” group was performed using one-wayANOVA with Holm-Sidak's multiple comparison test. Both the GM-CT-01 andthe GA-MD-02 compounds were shown to reduce lung hydroxyproline contentcompared to vehicle-treated control mice.

The Modified Ashcroft Index score was determined for the vehicle-treatedcontrol mice and the GR-MD-02 and GM-CT-01 treated mice. FIG. 4 showsthe Modified Ashcroft Index score with outliers removed. There were twonotable outliers. One vehicle-treated control mice was essentially notaffected by bleomycin administration. In the GR treatment group, therewas one animal with severe fibrosis. If these two animals are removedfrom the analysis, there is a significant different between vehicle andGR-MD-02 treated animals (p=0.0112, unpaired t-test). There is also asignificant difference between mice and GM-CT-01 treated animals(p=0.0199, unpaired t-test).

The general fibrosis score was determined. FIG. 5 shows the generalfibrosis score with outliers removed. There were two notable outliers.One vehicle-treated control mice was essentially not affected bybleomycin administration. In the GR-MD-02 treatment group, there was oneanimal with severe fibrosis. If these two animals are removed from theanalysis, there is a significant different between vehicle and GR-MD-02treated animals (p=0.0206, unpaired t-test) and GM-CT-01 treatmentapproaches significance (p=0.0979, unpaired t-test).

Inflammation score was determined. FIG. 6 shows the Inflammation scorewith outliers removed. There were two notable outliers. Onevehicle-treated control mice was essentially not affected by bleomycinadministration. In the GR-MD-02 treatment group, there was one animalwith severe fibrosis. If these two animals are removed from theanalysis, then both treatment approach significance (GM-CT-01 vs.vehicle, p=0.0544 unpaired t-test and GR-MD-02 vs. vehicle, p=0.0508,unpaired t-test).

Lung pathology with trichrome stain of saline control,vehicle+bleomycin, GM-CT-01 and GR-MD-02 treated animals was examined.All tissue samples were examined and examples are shown in FIG. 7. BothGM-CT-01 and GR-MD-02 treatment were found to be effective at reducingfibrosis. Both the number of animals with fibrosis and the averagedegree of fibrosis were reduced with both treatments. Vehicle-treatedanimals tended to have contiguous areas of fibrosis affecting largeareas of most lung lobes. Fibrous connective tissue stains blue withtrichrome and is visualized as steaming blue fibers (asterisks,vehicle-treated animal, FIG. 7). With both treatments, animals hadsmaller areas of fibrosis. There were often small knot-like formations(arrows, GR-MD-02 treated animal, FIG. 7) or small masses (thick arrows,GM-CT-01 treated animal, FIG. 7) of fibrosis, but these tended not toaggregate to the large formation seen in the vehicle-treated animals andto affect a smaller percentage of lungs.

Representative images with H & E are shown in FIG. 8. H & E images showthe same basic features as those stained with the trichrome stain. Thereare mixed mononuclear cell infiltrate associated with the fibrosis. Thisinflammation is more clearly evident with the HE stain (arrows, FIG. 8).

The foregoing results showed that treatment with GM (GM-CT-01) and GA-RG(GR-MD-02) caused a significant reduction in mean lung weight and lunghydroxyproline content compared to the vehicle-treated control group.

Histologic analysis indicated that GM-CT-01 and GR-MD-02 produced asignificant reduction in the Modified Ashcroft Index Score compared tothe vehicle group.

Although the rest of the histological findings were not statisticallysignificant, there were clear, notable trends suggesting a positivetreatment effect for both drugs.

The results with the GM compound (GM-CT-01) and the GA-RG compound(GR-MD-02) are superior to the results using pirfenadone which is asmall molecule comprising a modified phenyl pyridine. The results withGR-MD-02 were found to be similar to treatment with anti-TGFbeta, andthe results with GM-CT-01 were found to be superior to treatment withanti-TGFbeta in the experience of the laboratory.

Example 2 Late Treatment of Pulmonary Fibrosis Induced by Bleomycin inC57BI/6 Mice

In these experiments, treatment was initiated after the time when theinflammatory reaction is known to have subsided and the process is oneof primarily fibrogenesis. Pulmonary fibrosis was induced in animalsusing bleomycin at 2.25 U/kg at day 0 as described above. Animals weretreated using a GM compound intravenously (GM-CT-01) or GA-RG compoundintravenously (GR-MD-02) as described in Table 2.

TABLE 2 Bleomycin Test Article Group Number of Dose Dosing NumberAnimals (Day 0) Treatment Route Schedule 1 10 Saline — — — 2 12 2.25U/kg Vehicle IV QD Days: 10, 13, 16, 20 3 12 2.25 U/kg GM-CT-01 IV QD120 mg/kg Days: 10, 13, 16, 20 5 12 2.25 U/kg GR-MD-02 IV QD  60 mg/kgDays: 10, 13, 16, 20

Animals were euthanized on Day 21 and the lungs were weighed. Data shownin FIG. 9 represent group means and standard error of the means. Whilethere was a decreased lung weight of the GR-MD-02 treated group ascompared to control animals, this difference did not reach statisticalsignificance.

The total lung hydroxyproline content was measured. Data shown in FIG.10 represent group mean and expressed as microgram of hydroxyproline permg of lung tissue. Treatment with GR-MD-02 reduced the increase inhydroxyproline by 50%, but because of the variability in the data, thisdifference did not reach significance. There was less of a decrease inGM-CT-01 animals.

The Modified Ashcroft Index score was determined for the vehicle-treatedcontrol mice and the GR-MD-02 and GM-CT-0-treated mice. FIG. 11 showsthe Aschcroft score. There was a reduction in the Aschroft score in theGR-MD-02 treated animals as compared to vehicle treated animals, but thechange did not reach significance. There was little change in theGM-CT-01 treated group.

FIG. 12 shows the general fibrosis score for each animal group. Therewas a reduction in the fibrosis score in animals treated with bothGM-CT-01 and GR-MD-02 as compared to the vehicle-treated animals, butthe differences did not reach statistical significance.

FIG. 13 shows the Inflammation score for each animal group. There was areduction in inflammation score in the group treated with GR-MD-02 ascompared to vehicle controls, but no change in the GM-CT-01 group ascompared to vehicle control. The differences seen were not statisticallysignificant.

Lung pathology with trichrome stain of saline control,vehicle+bleomycin, GM-CT-01 and GR-MD-02 treated animals was examined.All tissue samples were examined and examples are shown in FIG. 14.GR-MD-02 treatment was found to be effective at reducing fibrosis, whileGM-CT-01 had less effect. Vehicle-treated animals tended to havecontiguous areas of fibrosis affecting large areas of most lung lobes.GR-MD-02 treated animals had smaller areas of fibrosis. There were oftensmall knot-like formations or small masses of fibrosis, but these tendednot to aggregate to the large formation seen in the vehicle-treatedanimals and to affect a smaller percentage of lungs.

Representative images with H & E are shown in FIG. 15. H & E images showthe same basic features as those stained with the trichrome stain. Thevehicle control animals showed areas of dense fibrosis. There was somereduction in the fibrosis in the GM-CT-01 treated animals as indicatedby more air space in the regions of fibrosis. In the GR-MD-02 treatedanimals there was a marked decrease in fibrous tissue.

Under the conditions of this study, both GM-CT-01 and GR-MD-02 treatmentwere effective at reducing pulmonary fibrosis in the bleomycin inducedpulmonary fibrosis mouse model.

The results showed that GR-MD-02 exhibited the strongest anifibroticeffect under the conditions used. Animals in this group tended to havereduced percent of fibrotic lung, inflammation, semi quantitativegeneral fibrosis score and fibrosis index score. While these changeswere not statistically significant, they were noticeable and there wereclear trends in the data supportive of a positive treatment effect.

The results showed that GM-CT-01 exhibited a more modest positivetreatment effect. The percent of lung affected and semiquantitativefibrosis score were notably reduced but these findings were notstatistically significant. There was less improvement in the index andinflammation scores. Regardless, there were clear trends in the datasupportive of a positive treatment effect.

Conclusions

In the early treatment model, both GR-MD-02 and GM-CT-01 were shown tomarkedly reduce lung weight and hydroxyproline content, with reductionof histological evidence of inflammation and fibrosis when compared tovehicle-treated bleomycin mouse model.

Treatment: results showed that GR-MD-02 was more effective than GM-CT-01in treating pulmonary fibrosis in the mouse model.

A mouse chronic model, such as intravenous bleomycin, can be used infuture studies to evaluate the effect of different dosages and/ordifferent schedules of administration.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the invention. Allpublications, patents and sequence database entries mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

1. A method comprising: a. obtaining a composition for parenteral orenteral administration comprising a galacto-rhamnogalacturonate in apharmaceutical acceptable carrier, wherein thegalacto-rhamnogalacturonate comprises a 1,4-linked galacturonic acid(GalA) and methyl galacturonate (MeGalA) residues backbone linked tobranched heteropolymers of alternating oligomers of α-1,2 linkedrhamnose and α-1,4-linked GalA residues, the rhamnose residues carryinga primary branching of oligomers of 1,4-β-D-galactose residues,1,5-α-L-arabinose residues, or combinations thereof, and wherein the1,4-β-D-galactose and 1,5-α-L-arabinose residues are present in a 2:1 toa 3:1 ratio, and b. administering to a subject in need thereof aneffective dose of the composition that results in at least 5%improvement of pulmonary function tests, wherein the subject in needthereof has at least one of the following: a primary or secondary lungfibrotic disease.
 2. The method of claim 1 wherein the step ofadministering results in at least 5% improvement of forced vitalcapacities or oxygen diffusion.
 3. The method of claim 1 wherein theeffective dose is equivalent to an animal dose of 10 mg/kg to 180 mg/kggiven once, twice or three times weekly.
 4. The method of claim 1wherein the pro-inflammatory proteins comprise TGF-beta, IL-6, IL-8,IL-13, osteopontin, TNF-alpha, CXCL-9/10, or VEGF.
 5. The method ofclaim 1 wherein the fibrogenic proteins comprise collagen, or elastin.6. The method of claim 1 wherein the galacto-rhamnogalacturonate furthercomprises xylose, glucose, fucose residues or combination thereof. 7.The method of claim 1 wherein the galacto-rhamnogalacturonate issubstantially free of 1,5-α-L-Ara residues.
 8. The method of claim 1wherein the 1,4-β-D-galactose residues, the 1,5-α-L-arabinose residuesor combination thereof represent at least 10 molar percent of the totalmolar carbohydrates.
 9. The method of claim 1 wherein thegalacto-rhamnogalacturonate has an average molecular weight ranging from5 kDa to 55 kDa as determined by SEC-RI and/or the SEC-MALLS methods.10. The method of claim 1 wherein the galacto-rhamnogalacturonate has anaverage molecular weight ranging from 2 kDa to 80 kDa as determined bySEC-RI and/or the SEC-MALLS methods.
 11. The method of claim 1 whereinthe galacto-rhamnogalacturonate has an average molecular weight rangingfrom 20 kDa to 70 kDa as determined by SEC-RI and/or the SEC-MALLSmethods.
 12. A method comprising: a. obtaining a composition forparenteral or enteral administration comprising a galactomannanpolysaccharide in a pharmaceutical acceptable carrier, and b.administering to a subject in need thereof an effective dose of thecomposition that results in at least one of the following: at least 5%reduction of lung edema, at least 5% reduction of lung pathologyseverity scores associated with lung fibrosis, at least 5% reduction oflung tissue hydroxyproline accumulation, at least 5% reduction ofexpression of pro-inflammatory proteins, at least 5% reduction ofexpression of fibrogenic proteins, at least 5% improvement of pulmonaryfunction tests, wherein the subject in need thereof has at least one ofthe following: a primary or secondary lung fibrotic disease.
 13. Themethod of claim 12 wherein the step of administering results in at least5% improvement of forced vital capacities or oxygen diffusion.
 14. Themethod of claim 12 wherein the effective dose is equivalent to an animaldose of about 60 mg/kg.
 15. The method of claim 12 wherein in the stepof obtaining, the galactomannan polysaccharide has a mannose togalactose ratio ranging from 1:1 to 1:4.
 16. The method of claim 12wherein in the step of obtaining, the galactomannan polysaccharide has amannose to galactose ratio of 1.7:1.
 17. The method of claim 12 whereinin the step of obtaining, the galactomannan polysaccharide has amolecular weight of about 48,000 D.
 18. The method of claim 12 whereinin the step of obtaining, the galactomannan polysaccharide has anaverage molecular weight ranging from 4,000 D to 60,000 D.